Using probes generated by chromosome microdissection and fluorescence in situ hybridization, we have identified a series of chromosomal regions involved in amplification events not previously recognized in human breast, ovarian and prostate cancer. In addition, it has been possible to determine the complex structure of homogeneously staining regions in several cases establishing that these structures are frequently composed of DNA segments derived from multiple chromosomes and intermingled to form an abnormal region. In order to isolate candidate target genes from these regions, we have developed a technique based on microdissection-mediated hybrid-selection of cDNAs. This technology, which supplements standard positional cloning techniques, has been utilized to isolate candidate genes from amplified regions on chromosome 13, 17 and 20 in breast cancer, as well as chromosome 12 in sarcomas. In addition, microdissection probes have been used to evaluate clinical material for the presence of gene amplification by FISH in prostate cancer and sarcomas. The methodology previously described illustrates the importance of developing rapid techniques for the identification of genes amplified in a series of key human tumors. In addition to recognizing known sites of gene amplification, we have identified several previously unidentified genes amplified in breast, prostate and ovarian cancers. These genes will be utilized to determine clinical significance of gene amplification in various malignancies. It appears highly likely that important new information will be discovered relative to genes playing a causal role in disease genesis or progression.